The synthesis of ribosomes in bacteria is regulated in response to changes in the environment. Our goal is to understand the molecular details of the mechanisms underlying this regulation. The information gained by this study should contribute to our understanding of the molecular basis for regulation of cell growth. The genes for Escherichia coli ribosomal proteins (r-proteins) are organized into about 20 operons. The r-protein operons are subject to at least two types of regulation: a) modulation of transcription initiation, and b) operon-specific autogenous regulation, whereby unique r-proteins repress their own operons. Our recent results suggest that additional regulatory processes also affect r-protein synthesis. We are investigating the mechanisms regulating the eleven gene S10 r-protein operon. 1. Autogenous regulation of the operon is mediated by r-protein L4. In the presence of excess L4, transcription is terminated prematurely at an attenuator site in the leader. Based on genetic results, we have designed a model for this process. To refine this model we will: a) use site-directed mutagenesis to obtain more regulatory mutants; b) determine the secondary structure of the leader of the S10 mRNA; c) analyze the binding of L4 to the S10 leader; d) establish an in vitro system for biochemical studies of the attenuation process. 2. To characterize the translation and degradation of mRNA from the S10 operon, we will a) use site-directed mutagenesis and studies of mRNA structure to determine the molecular basis for coupled translation of the most proximal two genes; b) investigate if downstream genes are subject to coupled translation; c) identify the sequences necessary for efficient translation of the first gene of the operon; d) study the role of message stability in regulation of gene expression. 3. To study regulation at the S10 promoter, we will isolate and characterize mutations in the upstream region, the -35 and -10 regions, and the stringent discriminator region. 4. To identify other mechanisms involved in regulating ribosome synthesis, we will follow up on preliminary indications that there are targets for growth rate regulation outside the proximal part of the operon. Our experiments indicate that several regulatory processes contribute to the regulation of r-protein synthesis in response to a given physiological condition. We will continue investigations of the interactions between these different regulatory mechanisms.